Over expression of recombinant gene products in the periplasm of Escherichia coli results is frequently associated with unfolded or misfolded protein and may involve degradation by cellular proteases. Furthermore, uncontrolled leakage or lysis of cells, caused by misfolding can inhibit fermentation processes directly or by an overflow of foam. Therefore, several strategies have been developed to improve the expression and folding properties of proteins in the periplasm of Escherichia coli. At first, a successful expression and folding of recombinant proteins is closely linked to the choice of optimal regulatory sequences, e.g. promoter strength, ribosome binding sites and signal peptides1-3. Application of folding strategies mainly refer to feeding of folding promoting agents4-10, to the coexpression of molecular chaperones and to adding folding catalysts11-15.
Folding promoting agents such as glycine, betaine and hydroxyectoine are known protein protectants in the art48-50. In general it is believed, that these compounds do not strengthen the protein conformation by specific binding as would a substrate or an inhibitor. The stabilizing effect of these compounds has been attributed mainly to their exclusion from the protein surface, hence leading to ‘preferential hydration’ of the protein, or ‘preferential exclusion’ of the additive from the protein surface. However the stabilizing phenomenon is a rather complex one, and it has to be pointed out, that there is no single mechanism responsible for the stabilization but a multitude of stabilizing and destabilizing interactions besides the preferential exclusion mechanism.
In addition to using extrinsic folding promoting agents the protein itself can be improved either by molecular modeling or directed evolution, here and elsewhere experiments performed have used scFv antibody fragments16-19.
It has to be pointed out that none of all the individual strategies is generally successful and therefore the folding of proteins has to be improved sequentially and case by case. This requires technologies for direct folding monitoring, which ideally are independent of functional assays. The current invention delivers the technical solution to this problem. In contrast to recent works20-21 the invention does use the native stress response to misfolded protein in the periplasm of Escherichia coli, regulated by two partially overlapping pathways, the sigma E response and the Cpx signal transduction system24. Sigma E is tightly regulated by three genes, rseA, rseB and rseC25. The transmembrane protein RseA senses and transmits information to sigma E, negatively regulated by the interaction with the periplasmic RseB and positively by RseC, respectively, located in the inner membrane. The Cpx two-component signal transduction system consists of a membrane sensor histidine kinase CpxA and a cytoplasmic response regulator CpxR. Misfolded protein leads to autophosphorylation of CpxA followed by a phosphotransfer to CpxR, allowing CpxR to function as a transcriptional activator27-29. Both the sigma E and the Cpx response induce several genes involved in protein folding and degradation in the case of periplasmic misfolding. The Cpx signal transduction system coordinates the activation of DsbA, PpiA and PpiD20-30, whereas sigma E regulates the transcription of at least 10 gene products, including even sigma E, sigma 32 and fkpA31-35. Only degP (htrA) is regulated by both systems, indicating that degP is a central element in the periplasmic misfolding management.36-40. In this respect, the invention among other aspects demonstrates that a degP promoter based reporter system is very suitable for kinetic studies of protein misfolding in the periplasm of Escherichia coli and allows an effective use in combination with different protein folding strategies.